The objective of this research is to study evolutionary forces that govern unusual variation of the D. melanogaster fourth chromosome uncovered recently by us. For a long time, this chromosome has been viewed as a canonical textbook example of how natural selection can result in low within-species variation in a genome region of low recombination. Our preliminary work has indicated that the previous conclusion that the entire chromosome 4 harbors low variation is incorrect and should not be taken as a paradigm any more. We have found a high level of nucleotide variation in large blocks of this chromosome in D. melanogaster and simulans. The following objectives will be pursued. (1) Using Monte Carlo simulations based on the coalescent process with selection and recombination, we will investigate a theoretical population genetic process associated with a low average recombination rate in the whole chromosome but with sporadic recombination hot spots. We expect that this theoretical analysis will shed new light on the relationship between recombination and variation. (2) To determine the length of the high variation region in the chromosome, we will characterize the polymorphism distribution in natural populations in whole euchromatin regions of the D. melanogaster fourth chromosome. (3) To compare the high variation region of D. melanogaster with the corresponding region in other sibling species, we will investigate sequence variation in D. simulans and D. yakuba. Finally, using these theoretical results and variation data, we will test several hypotheses that might explain the variation and evolution of the chromosome: selective sweep, background selection, balancing selection, and neutrality. We will also characterize the variation and evolution of the sphinx gene, a gene that recently originated on the D. melanogaster fourth chromosome, with structural and expression analysis, and investigate its potential role in the evolution of the chromosome. These studies should add significantly to the understanding of the evolutionary genetics of the Drosophila fourth chromosome.